We propose to carry out a comprehensive and comparative structure-function analysis of the alphaviruses and flaviviruses. The proposed research will investigate all of the structural and nonstructural proteins of these viruses, taking advantage of rapidly developing high-throughput technologies. A detailed understanding of these viral components and their role in the virus life cycle will be invaluable for the development of effective antiviral strategies as well as providing insight into basic macromolecular processes. The flaviviruses and alphaviruses comprise two groups of enveloped plus-strand RNA viruses with members that constitute significant threats to human health and potential biological weapons. The alphaviruses include pathogens such as Sindbis, Semliki Forest, and Venezuelan equine encephalitis virus, while the flaviviruses include yellow fever, dengue, West Nile, and tick-borne encephalitis virus. We will use molecular genetics, biochemistry, and structural techniques to probe the role of specific viral proteins in virus entry, protein processing, RNA replication, virus assembly, and virus budding. In collaboration with Tim Baker, Jue Chen, Michael Rossmann, and Jim Strauss, we will use cryo-electron microscopy and X-ray crystallography to examine the structure of the viruses and their component proteins, virus intermediates, and virus receptor or antibody complexes. Information obtained from these studies will guide molecular genetic experiments probing functional aspects of virus structure, assembly, and entry. We will work with Jue Chen, Janet Smith, and Jim Strauss to express and purify the viral nonstructural proteins for the purpose of biochemical and X-ray crystallographic studies. The atomic structures of these proteins will serve as a guide for mutagenesis experiments that will probe the relationships between their structures and their functions. Furthermore, comparative analyses between identical proteins from related viruses will advance our knowledge of virus evolution and protein function, providing novel insights into virus replication and pathogenesis.